We are investigating the subcellular location of hydrogen peroxide production in fibroblasts exposed to blue light (450-490 nm). Fluorescence microscopy (using the peroxide indicator dichlorofluorescein) has revealed generation of hydrogen peroxide from peroxisomes and mitochondria in 3T3 cells, but the fluorophore also responds to blue light which prevents/impedes quantitation of H202- We are proposing to use 2-photon microscopy so as to excite the fluorophore with infrared illumination and therefore avoid the production of peroxide from this source. This will enable us to separate the illumination used for peroxide production from the illumination used to excite the indicator fluorophore, thereby enabling quantitation of light-induced peroxide production by dichlorofluorescein.